Plasmodesmal targeting and intercellular movement of potato mop-top pomovirus is mediated by a membrane anchored tyrosine-based motif on the lumenal side of the endoplasmic reticulum and the C-terminal transmembrane domain in the TGB3 movement protein.

Live-cell fluorescence microscopy was used to investigate the third triple gene block protein (TGB3) of potato mop-top pomovirus and its role in assisted targeting of TGB2 to plasmodesmata (PD). Wild-type and mutant TGB3 proteins were expressed under the control of the 35S promoter or from a virus reporter clone. Assisted targeting of TGB2 to PD was optimal when the proteins were expressed from a bicistronic plasmid in the relative ratios expected in a virus infection, suggesting that excess TGB3 inhibited PD localisation. Contrary to the generally accepted view, bimolecular fluorescence complementation showed that the TGB3 N terminus is located in the cytosol. Mutational analysis to dissect TGB3 sub domain functions showed that PD targeting was mediated by a composite signal comprising an ER-lumenal tyrosine-based motif and the C-terminal transmembrane domain. Mutation of either of these domains also abolished cell-to-cell movement of the virus. The results are discussed in the context of TGB3 membrane topology.

Splicing signals and factors in plant intron removal.

Constitutive splicing of the potato invertase mini-exon 2 (9 nt long) requires a branchpoint sequence positioned around 50 nt upstream of the 5' splice site of the adjacent intron and a U(11) element found just downstream of the branchpoint in the upstream intron [Simpson, Hedley, Watters, Clark, McQuade, Machray and Brown (2000) RNA 6, 422-433]. The sensitivity of this in vivo plant splicing system has been used to demonstrate exon scanning in plants, and to characterize plant intronic elements, such as branchpoint and poly-pyrimidine tract sequences. Plant introns differ from their vertebrate and yeast counterparts in being UA- or U-rich (up to 85% UA). One of the key differences in splicing between plants and other eukaryotes lies in early intron recognition, which is thought to be mediated by UA-binding proteins. We are adopting three approaches to studying the RNA-protein interactions in plant splicing. First, overexpression of plant splicing factors and, in particular, UA-binding proteins, in conjunction with a range of mini-exon mutants. Secondly, the sequences of around 65% of vertebrate and yeast splicing factors have high-quality matches to Arabidopsis proteins, opening the door to identification and analysis of gene knockouts. Finally, to discover plant-specific proteins involved in splicing and in, for example, rRNA or small nuclear RNA processing, green fluorescent protein-cDNA fusion libraries in viral vectors are being screened.

Identification of the pharmaceutical care issues of rheumatoid arthritis patients in secondary care.

OBJECTIVE: To explore the perceptions of multidisciplinary health care professionals (HCPs) and patients of the pharmaceutical care issues (PCIs) relating to rheumatoid arthritis (RA). DESIGN: Qualitative study using semi-structured one to one interviews and focus groups to explore patient perceptions. Interviews and focus groups were taped and transcribed verbatim, then described and coded for meaning to produce 'in-vivo' codes, which were then grouped to form themes. Nominal group methodology was used to generate and rank a list of HCP perceptions of the key PCIs of RA patients. The PCIs were ranked according to clinical importance and order of occurrence from admission as perceived by the HCP group. SETTING: Rheumatology ward and outpatient clinic in a teaching hospital. MAIN OUTCOME MEASURES: Generation and ranking of PCIs, generation of themes from patient interviews. RESULTS: Optimisation of pain control was identified by the nominal group as being the primary aim for patients on admission and was also the most commonly described symptom by patients. Two PCIs not predicted by the HCPs' nominal group was the frequency of infections and the associated discharge and patients described experiencing 'over-education' by HCPs, which could lead to anxiety. Complementary medicine in conjunction with traditional therapy was raised as a significant health benefit by patients. CONCLUSION: Many patients' views mirrored the PCIs identified by HCPs, but some were not anticipated; the value of patient interviews to ensure appropriate service development was demonstrated. Several PCIs emerged for future incorporation by the multi-disciplinary team into standardised models of pharmaceutical care for use in secondary care and at the secondary/primary care interface for improvement of seamless care. There is a need to target educational interventions and to identify those who will benefit from advice on complementary medicine. Further work is required to develop a tool to identify the educational needs of RA patients and targeting of the information provided. This will help ensure the delivery of pharmaceutical care is designed to match the needs of individual patients.

Soil phosphorus variability in pastures: implications for sampling and environmental management strategies.

Soil phosphorus (P) is an increasingly important consideration in the development of P-based nutrient management strategies. The objectives of this study were to (i) obtain baseline information on soil P variability in pastures amended with animal waste, (ii) examine if current sampling recommendations related to the number of subsamples adequately reduce uncertainty to acceptable limits, and (iii) examine the implications of uncertainty in soil P estimates on implementing a soil P threshold of 150 mg kg(-1). Grid soil samples were collected from 12 pastures. Soil P was determined using Mehlich 3 extractant and an inductively coupled argon plasma spectrometer. The arithmetic mean of soil P ranged from 7 mg kg(-1) in a pasture never amended with animal manure to 437 mg kg(-1) in a pasture that had been annually treated long term with poultry litter. Variance of soil P generally increased with mean soil P. The mean standard deviation of all pastures was one-third of the 150 mg kg(-1) threshold. This study points out that smaller variances associated with mean soil P values that approach, but do not exceed, the threshold can influence estimates of soil P. In turn, management decisions could inappropriately change. When a uniform acceptance criteria (within 15 mg kg(-1)) with respect to measured means was used, the required minimum number of subsamples increased with measured standard deviation. The results of this study imply that following soil-sampling recommendations is critical to obtaining trustworthy measures of central tendency, especially in pastures approaching but not exceeding the 150 mg kg(-1) threshold.

The effects of the acetylcholinesterase inhibitor ENA713 and the M1 agonist AF150(S) on apolipoprotein E deficient mice.

Apolipoprotein E (apoE)-deficient and control mice were treated chronically with either the acetylcholinesterase (AChE) inhibitor ENA713, or the M1 muscarinic agonist AF150(S). Both treatments reversed the spatial working memory impairment of apoE-deficient mice but they differed in their effects on the levels of brain AChE activity. AF150(S) enhanced the brain AChE activity of apoE-deficient mice and rendered it similar to that of the untreated controls, whereas ENA713 reduced the brain AChE activity of control mice but had no effect on that of apoE-deficient mice. These findings suggest that AChE inhibition and M1 muscarinic activation have similar beneficial cognitive effects on apoE-deficient mice, but that the cellular and molecular mechanisms underlying these effects differ.

Specific neurochemical derangements of brain projecting neurons in apolipoprotein E-deficient mice.

Apolipoprotein E (apoE)-deficient mice provide a useful system for studying the role of apoE in neuronal maintenance and repair. Previous studies revealed specific memory impairments in these mice that are associated with presynaptic derangements in projecting forebrain cholinergic neurons. In the present study we examined whether dopaminergic, noradrenergic, and serotonergic projecting pathways of apoE-deficient mice are also affected and investigated the mechanisms that render them susceptible. The densities of nerve terminals of forebrain cholinergic projections were monitored histochemically by measurements of acetylcholinesterase activity, whereas those of the dopaminergic nigrostriatal pathway, the noradrenergic locus coeruleus cortical projection, and the raphe-cortical serotonergic tract were measured autoradiographically using radioligands that bind specifically to the respective presynaptic transporters of these neuronal tracts. The results obtained revealed that synaptic densities of cholinergic, noradrenergic, and serotonergic projections in specific brain regions of apoE-deficient mice are markedly lower than those of controls. Furthermore, the extent of presynaptic derangement within each of these tracts was found to be more pronounced the further away the nerve terminal is from its cell body. In contrast, the nerve terminal density of the dopaminergic neurons that project from the substantia nigra to the striatum was unaffected and was similar to that of the controls. The rank order of these presynaptic derangements at comparable distances from the respective cell bodies was found to be septohippocampal cholinergic > nucleus basalis cholinergic > locus coeruleus adrenergic > raphe serotonergic > nigrostriatal dopaminergic, which interestingly is similar to that observed in Alzheimer's disease. These results suggest that two complementary factors determine the susceptibility of brain projecting neurons to apoE deficiency: pathway-specific differences and the distance of the nerve terminals from their cell body.

Podophyllin 0.5% or 2.0% v podophyllotoxin 0.5% for the self treatment of penile warts: a double blind randomised study.

OBJECTIVE: To compare the effectiveness and cost of self treatment of penile warts with a commercial preparation of podophyllotoxin 0.5% (PDX 0.5%) with podophyllin 0.5% and podophyllin 2.0% sourced from Podophyllum emodii. DESIGN: A prospective double blind randomised study. SUBJECTS: 315 patients with penile warts attending two departments of genitourinary medicine. MAIN OUTCOME MEASURES: Absence of warts, cessation of treatment due to severe side effects at 5 weeks. RESULTS: Of the 315 patients, 244 conformed to the protocol. Analysis was on an intention to treat basis. At 5 weeks no significant differences were found in the extent of healing of warts or in side effects for the three treatment groups. The costs of drug treatment (excluding staff time) are at least pounds 10.00 less for podophyllin than podophyllotoxin. A fourfold variation in the active constituents of the podophyllin preparations did not produce appreciably different clinical responses. In a subanalysis no evidence of deterioration in effectiveness of podophyllin over time was demonstrated. CONCLUSIONS: Penile warts in selected cases can be safely treated with 0.5-2.0% podophyllin self applied by the patient at a fraction of the cost of commercially available podophyllotoxin. The shelf life of the podophyllin extracts is at least 3 months. These findings may be especially relevant in countries where resources for health care are limited.

Soil and solid poultry waste nutrient management and water quality.

Concerns about the impacts of nitrogen, phosphorus, and pathogens on surface and ground water quality has forced the poultry industry to implement voluntary waste management guidelines for use by growers. In some states, animal waste guidelines are being enforced by regulatory agencies. Strategies that growers may use to properly dispose of poultry waste include: 1) local land application as a fertilizer; 2) offsite marketing for use as a fertilizer or soil amendment, feed additive, or energy source; and 3) chemical additives that will immobilize nitrogen and phosphorus in the manure or litter. If properly followed, these and other innovative strategies should be adequate to protect surface and ground water quality without adversely affecting the economics of poultry production.

Jellyfish green fluorescent protein as a reporter for virus infections.

The gene encoding green fluorescent protein (GFP) of Aequorea victoria was introduced into the expression cassette of a virus vector based on potato virus X (PVX). Host plants of PVX inoculated with PVX.GFP became systemically infected. Production of GFP in these plants was detected initially between 1 and 2 days postinoculation by the presence of regions on the inoculated leaf that fluoresced bright green under UV light. Subsequently, this green fluorescence was evident in systemically infected tissue. The fluorescence could be detected by several methods. The simplest of these was by looking at the UV-illuminated plants in a darkened room. The PVX.GFP-infected tissue has been analysed either by epifluorescence or confocal laser scanning microscopy. These microscopical methods allow the presence of the virus to be localized to individual infected cells. It was also possible to detect the green fluorescence by spectroscopy or by electrophoresis of extracts from infected plants. To illustrate the potential application of this reporter gene in virological studies a derivative of PVX.GFP was constructed in which the coat protein gene of PVX was replaced by GFP. Confocal laser scanning microscopy of the inoculated tissue showed that the virus was restricted to the inoculated cells thereby confirming earlier speculation that the PVX coat protein is essential for cell-to-cell movement. It is likely that GFP will be useful as a reporter gene in transgenic plants as well as in virus-infected tissue.

Mutational analysis of the coat protein gene of potato virus X: effects on virion morphology and viral pathogenicity.

The role of the coat protein of potato virus X (PVX) was investigated by site-directed mutation of the coat protein gene. Mutant viruses with in-frame deletions in the 5' end of the coat protein gene were capable of systemically infecting plants, but produced virions with atypical morphology. Viruses with a frameshift mutation near the 5' end or with deletions in the central part of the coat protein gene failed to accumulate at detectable levels, even in the inoculated leaf. In protoplasts, mutants that infected systemically either had a wild-type phenotype or showed a small reduction in accumulation of genomic RNA. The other mutants, which did not accumulate in the inoculated leaf, were unaffected in genomic RNA accumulation 8 hr postinoculation, but at 16 hr and later they accumulated less genomic RNA than wild-type virus. None of the mutations had an effect on accumulation of negative-strand RNA. The data indicate that efficient accumulation and spread of PVX, even in the inoculated leaf, requires coat protein production and encapsidation of the viral RNA.

Molecular analysis of a resistance-breaking strain of potato virus X.

Full-length cDNA clones of potato virus X (PVX) strains PVXUK3 and PVXHB have been constructed in plasmid vectors to allow in vitro transcription of infectious PVX RNA. In both instances the transcript-derived virus infected tobacco and potato identically to the respective progenitor strains: in tobacco and susceptible potato cultivars both strains infected systemically, producing symptomless or mild mosaic symptoms. In potato carrying the Rx or Nx resistance genes, the virus derived from the PVXHB cDNA infected systemically, whereas the virus derived from the PVXUK3 cDNA failed to infect the Rx plants or induced apical necrosis, characteristic of a hypersensitive response of the Nx gene. Three hybrid viral genomes were constructed at the cDNA level to localize the resistance breaking determinants of PVXHB. Transcripts of all three hybrids were infectious on tobacco. On potato cultivars with either the Rx or Nx resistance genes, the hybrid viruses infected in the same way as PVXHB, rather than PVXUK3. The common feature of these hybrid viruses, the coat protein gene, is therefore the determinant of Nx and Rx resistance breaking of PVXHB.

A fully differentiating epidermal model with extended viability: development and partial characterization.

A highly differentiated porcine skin organ culture model has been developed for future investigations of membrane-coating granules (MCG) and their role in epidermal differentiation. In contrast to many previous systems, cultures do not undergo necrosis of the upper epidermis or display dermo-epidermal separation, but survive for at least 3 weeks, at which time mitotic cells are still evident. Although rete projections are gradually smoothed out and the viable epidermis thins at a rate of approximately 0.35 cells per day, the stratum corneum gains approximately 1.5 corneocytes per day. Furthermore, at 3 weeks all the major differentiation markers are expressed, including keratohyalin granules, MCG, and an orthokeratotic stratum corneum. The system is inexpensive, simple to establish, and does not require elevated oxygen levels. The main requirements are 1) the use of Dulbecco's minimal essential medium supplemented with 2) hydrocortisone (100 micrograms/ml), 3) growth at an air/liquid interface, and 4) attached connective tissue. The further addition of vitamin C (300 micrograms/ml) and/or bovine serum albumin (2 mg/ml) offered no obvious advantage. Degeneration of organ cultures in standard cell culture media was discovered to be caused by fetal bovine serum (FBS). FBS-induced degeneration was not prevented by adding any of the supplements tested, or the inclusion of 3T3 fibroblasts, even when culturing at an air/liquid interface. Complete submersion rapidly killed specimens, presumably through oxygen starvation. The ability to maintain a fully keratinizing system for several weeks, in a totally chemically defined medium, will prove valuable for research not only into the role(s) of MCG in epidermal biology but also studies of desquamation and epidermal differentiation.

Blistering in keratinocyte cultures: a regular phenomenon associated with differentiation.

Blisters have previously been observed in keratinocyte cultures depleted of vitamin A, and in cultures of keratinocytes from patients with epidermolysis bullosa. We have found that blistering may occur in keratinocyte cultures from normal human epidermis, grown under standard conditions, and our aim was to further characterize the mechanism of blister formation. Keratinocytes were seeded at 10(5) cells per 35 mm collagen-coated dish with a 3T3 feeder layer. Blisters were macroscopic, fluid-filled structures which formed irrespective of donor site, or donor age, and were noted on various alternative substrates (collagen, 3T3 + plastic, plastic alone). Blistering commenced around day 12, prior to confluency, and new blisters were formed for up to 5 weeks post-plating. Maximal numbers (up to 70 per dish) were present around days 12 to 20. Cleavage occurred at the cell/collagen interface to form a blister roof composed of 6 to 9 cell layers. The lowest layer appeared metabolically active, but, in contrast to peri-blister regions, lacked hemidesmosomes. The central 2 to 3 layers contained membrane-coating granules and keratohyalin granules while the superficial strata resembled rudimentary corneocytes. Cultures supplemented with 10(-5) M vitamin A formed no blisters, which correlated with suppressed differentiation. Ouabain (10(-7) M) caused blister collapse and a reversible inhibition of new blister formation. We conclude that blisters are a consistent finding in keratinocyte cultures grown under standard conditions. Their formation may be associated with active transport and triggered during differentiation. Further examination of this phenomenon might shed light on whether differentiation itself has an influence on keratinocyte attachment to substrate.

Staphylococcal beta-lactamase and efficacy of beta-lactam antibiotics: in vitro and in vivo evaluation.

The susceptibility of five cephalosporins and nafcillin to changes in inoculum size of 26 isolates of Staphylococcus aureus was studied. The spectrum of antibiotic sensitivity to such changes was cefazolin = cephaloridine greater than cefamandole greater than cephalothin greater than cefoxitin = nafcillin. Half of the isolates resulted in large and half in minimal inoculum-induced changes in minimal inhibitory concentrations (MICs). These changes correlated with the amount of beta-lactamase produced by the isolates. The in vivo relevance of these findings was studied in a model of intraperitoneal infection in mice. The effect of beta-lactamase production on mortality was greatest among animals given cefazolin or cephaloridine, intermediate among those given cefamandole, and nonexistent among those given cefoxitin, cephalothin, or nafcillin. The number of organisms in the animals' spleens paralleled survival rates. An increase in the serum level of cefazolin increased the survival rate among mice given that drug. Hence, the survival of mice was influenced by (1) the ability of the infecting organism to increase the MIC of an antibiotic via inoculum increases, (2) the sensitivity of the antibiotic to beta-lactamase, and (3) the peak level of antibiotic attained in serum.

Effect of vitamin D in fluoride-treated rats.

Fluoridated drinking water (30 mg and 100 mg F per liter) was used to induce rachitic changes in rats fed a vitamin D free diet containing calcium and phosphorus in a ratio of 1 : 1. Supplements of vitamin D3 (70 IU of cholecalciferol per week) completely prevented the rachitogenic effects of fluoride. This protective effect occurred despite evidence that vitamin D enhanced the intestinal absorption of fluoride.